Identification of pumpkin beetle (Aulacophora spp.) through mtDNA-COI barcode approach in cucurbitaceous vegetable crops

Cucurbitaceous vegetables are frequently attacked by various Aulacophora spp., making accurate species identification essential for effective pest management. However, traditional morphological methods have notable limitations in distinguishing these species. The present study was carried out during 2022 and 2023 at Bihar Agricultural University, Sabour, Bihar using molecular barcoding to provide a more reliable and precise identification approach. Genomic DNA was isolated from the mitochondria-rich legs and wings of 10 Aulacophora specimens collected from different agro-climatic zones of Bihar and the mitochondrial cytochrome c oxidase subunit I (COI) gene was amplified using universal primers. The PCR-amplified COI gene fragments were sequenced, and the resulting barcode sequences were deposited in the NCBI GenBank database, where accession numbers were assigned to all 10 entries. BLAST analysis of these sequences confirmed the presence of two species i.e. Aulacophora lewisii and Aulacophora indica through comparison with reference sequences from multiple countries available in GenBank. A phylogenetic tree constructed at a 0.020 bar scale grouped the sequences into three distinct clusters. Haplotype analysis indicated that A. lewisii and A. indica diverged from a common ancestor through specific mutational events. Furthermore, several highly expressed codons were detected in both species, which may influence protein synthesis and hold potential for future functional studies.